Profiling of Endogenously Intact N-Linked and O-Linked Glycopeptides from Human Serum Using an Integrated Platform
Profiling of Endogenously Intact N-Linked and O-Linked Glycopeptides from Human Serum Using an Integrated Platform
复制标题
使用集成平台对人血清中的内源完整 N-连接和 O-连接糖肽进行分析
DOI：
10.1021/acs.jproteome.9b00592
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发表时间：
2020
影响因子：
4.4
通讯作者：
Ye Mingliang
中科院分区：
文献类型：
--
作者：
Wang Shuyue;Qin Hongqiang;Mao Jiawei;Fang Zheng;Chen Yao;Zhang Xiaolei;Hu Lianghai;Ye Mingliang
血清中的内源性糖肽是发现生物标志物的宝贵资源。由于串联质谱的丰度低且碎片化差，内源完整糖肽的鉴定仍然面临许多挑战。在此，构建了一个用于鉴定 N-连接和 O-连接内源完整糖肽的集成平台。在该平台中，高温酸变性、超滤和亲水相互作用层析步骤结合在一起，可以从少量血清中高效提取内源完整糖肽。此外，双光谱方案和计算机去糖基化策略分别应用于 N 连接和 O 连接内源糖肽的鉴定。仅从 40 μL 人血清样品中就总共鉴定出了 223 种完整的 N-糖肽和 51 种完整的 O-糖肽。这是第一个报告鉴定内源完整N-连接和O-连接糖肽的研究，也是迄今为止报道的最大的内源完整糖肽数据集。进一步分析肽和蛋白质中聚糖的分布以及肽上的切割位点，以寻求内源糖基化对疾病机制的调节。所开发的策略为疾病生物标志物的发现提供了一个新的平台。
Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenously intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N-linked and O-linked endogenously intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and hydrophilic interaction chromatography steps are combined together for the highly efficient extraction of the endogenously intact glycopeptides from a small amount of serum. Additionally, the twin-spectra scheme and in silico deglycosylation strategy were applied for the identification of N-linked and O-linked endogenous glycopeptides, respectively. In total, 223 intactN-glycopeptides and 51 intactO-glycopeptides are identified from only 40 μL of the human serum sample. This is the first study reporting the identification of endogenously intact N-linked and O-linked glycopeptide and is also the largest data set of endogenously intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for the disease biomarker discovery.